1 Field of the Invention
The present invention relates generally to the detection of enzyme levels that are affected by a specific class of cancer chemotherapeutic agents, and more particularly, to the use of the enzyme spermidine/spermine N.sup.1 -acetyltransferase as a prognostic indicator and/or tumor response marker to facilitate clinical use of the class of anticancer agents that cause induction of the enzyme as a significant part of their biological effect profile.
2 Description of the Background and Related Art
The biological polyamines, putresine, spermidine and spermine are natural components of all mammalian cell types and are known to be essential for cell growth. While their precise role in supporting cell growth is uncertain, it is believed to involve interaction with nucleic acids. These substances and their key biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase (ODC and SAMDC, respectively), are increased in neoplastic tissues. Depletion of intracellular polyamine pools unequivocally inhibits cell growth under in-vitro conditions. Therefore, polyamine biosynthesis has been targeted in the development of experimental anticancer strategies.
Because inhibitors of polyamine biosynthetic enzymes lower the polyamine content of tumor cells, they are among the compounds being evaluated for use as anticancer agents. Thus far, these inhibitors have not lead to clinically effective anticancer agents. One of the problems confronting their use is that as soon as polyamine pools are lowered, the key biosynthetic enzymes ODC and SAMDC, as well as polyamine transport, undergo a compensatory increase in activity (Porter et al., 1992, in Polyamines in the Gastrointestinal Tract, pp. 301-322, Falk Symposium 62, (eds) Dowling et al., Academic Publishers). Thus, the desired effect of the enzyme inhibitors (i.e. inhibiting cell growth by polyamine pool depletion) is circumvented by one or both of these homeostatic responses.
Based on the observation that increases in the polyamine pools suppress ODC and SAMDC activities, an alternative approach has been devised (Porter and Bergeron, 1988, in Advances in Enzyme Regulation, pp. 57-79, Pergamon Press). It proposes to identify polyamine analogs which behave like the natural polyamines in down-regulating ODC and SAMDC, but which lack the ability to perform in functions required for cell growth. N.sup.1,N.sup.12 -bis(ethyl)spermine (BESPM), a N-bis(ethyl) analog of spermine, has served as a model compound for this strategy. A comparison of the chemical structures of spermine and BESPM is shown in FIG. 1. BESPM was found by in-vitro studies to rapidly suppress ODC and SAMDC, deplete natural polyamine pools, and inhibit cell growth at 1-10 uM (Porter et al., 1987, Cancer Res. 47:2821-2825). In addition, BESPM suppresses polyamine uptake (Byers and Pegg, 1990, J. Physiol. 142:460-467; and Kramer et al., 1993, J. Cell. Physiol. 115:399-407), and thus minimizes the ability of tumor cells to meet their polyamine requirement by taking them up from their environment. The potential for polyamine analogs as effective chemotherapeutic agents is evidenced by potent antitumor activity against several melanoma cell lines in-vitro (Porter et al., 1991, Cancer Res., 51:3715-3720; Shappell et al., 1992, Anticancer Res., 12:1083-1090) and correspondingly, in-vivo against MALME-3 and PANUT human melanoma tumors growing as xenografts in athymic mice treated with bis-ethyl spermine analogs (Bernacki et al., 1992, Cancer Res., 52:2424-2430; Porter et al., 1993, Cancer Res. 53:581-586). Potent antitumor activity of bis-ethyl spermine analogs has also been demonstrated for pancreatic cancer cell lines in-vitro (Chang et al., 1992, Cancer Chemother. Pharmacol. 30:183-188) and correspondingly in-vivo against mouse xenografts of human pancreatic ductal adenocarcinoma cells (Chang et al., 1992, Cancer Chemother. Pharmacol. 30:179-182). The bis-ethyl spermine analog with the most potent antitumor efficacy, N.sup.1,N.sup.11 -bis(ethyl)norspermine (BENSPM, also known as DENSPM) is the subject matter of a new drug application submitted to the FDA for approval of clinical trials at 3 research institutions.
Additional in-vitro studies show that BESPM, and related analogs also cause an profoundly large induction of the polyamine metabolizing enzyme spermidine/spermine N.sup.1 -acetyltransferase (SSAT) in certain human carcinoma cell lines. The following is a list of relevant prior art along with a brief description of each:
Libby et al. (Arch. Biochem. Biophys. 284: 238-244, 1991) and Casero et al. (Biochem. J., 270: 615-620) describe isolation and partial characterization of human SSAT protein from BESPM-treated cell lines. Casero et al. report that the cytotoxic response of the NCI H157 human large cell lung carcinoma cell line to exposure to BESPM was associated with a high induction of SSAT in-vitro.
Porter et al. (1991, Cancer Res., 51:3715-3720) describe the extreme induction of SSAT levels after treatment of human melanoma cell lines (MALME-3) in-vitro with BESPM and an even higher degree of induction with other bis-ethyl spermine analogs. Between two melanoma cell lines (LOX and MALME-3), which differentially induce SSAT activity, a correlation was established between enzyme induction and in-vitro growth sensitivity to the bis-ethyl spermine analogs.
Each of the above cited references discloses a high induction in the levels of SSAT after treatment of specific human solid tumor cell lines in-vitro with bis-ethyl spermine analogs. However, there is a need to establish a correlation between SSAT levels and tumor growth responses in animals. At the time of the invention, none of the references demonstrated the induction of SSAT levels following treatment of malignant solid tumor-bearing animals, i.e. in-vivo, nor did the references disclose correlation of malignant solid tumor growth sensitivity to bis-ethyl spermine analogs with induced SSAT levels in-vivo.
It is not known if the high. induction of SSAT would occur in-vivo because of uncertainties related to the stability of SSAT-specific m-RNA intracellularly and the stability of the enzyme itself. Because the analogs may cause other cellular events in in-vitro systems (i.e. suppression of ODC/SAMDC, depletion of polyamine pools, and possibly other DNA-related effects), it is important to demonstrate that SSAT induction in-vivo is prominent among those events and that it may be causally related to or indicative of antitumor activity. Because an event occurs in-vitro does not necessarily mean that the event occurs in-vivo. For instance, suppression of ODC and SAMDC is well recognized as an in-vitro effect of bis-ethyl spermine analogs. However, recent studies (Porter et al., 1993, Cancer Res. 53:581-586; Porter and Bergeron, 1988, supra) show that this effect does not occur in-vivo.
Moreover, it must be demonstrated that SSAT induction in-vivo occurs selectively, or to a greater degree in malignant solid tumor tissue relative to various normal tissues. Its potential usefulness as a tumor marker, and as a determinant of drug action, is highly dependent upon quantitatively selective induction of enzyme levels in tumor cells. At the time of the invention, the prior art discloses induction of enzyme in certain solid tumor cell lines in-vitro, but does not disclose relative enzyme levels in related normal tissue in-vivo.
Therefore, there exists a need for a sensitive biological response indicator for use in monitoring the clinical effectiveness of polyamine analog anticancer agents, including the bis-ethyl spermine analogs, which have potent anticancer activity against certain cancers comprising malignant solid tumors such as human melanoma, and human pancreatic adenocarcinoma, and which also induce SSAT activity. In addition, a method for evaluating therapeutic effectiveness of treatment, and for the sensitivity to treatment of an individual's tumor with analogs, such as a bis-ethyl spermine analog, is desired. Such methods will greatly facilitate the identification of, and chemotherapeutic treatment of individuals bearing malignant solid tumors sensitive to analogs that effect induction of SSAT such as bis-ethyl spermine analogs.